The present invention relates to a regulator protein involved in a plant two-component signal transduction system and to a nucleic acid coding for the same.
Cytokinins are a group of plant hormones, and have important roles in controlling growth, morphogenesis and flow of nutrients in plants. However, the mechanism by which a cytokinin is recognized by plant cells and causes various actions was not known. As a cytokinin, zeatin is well-known.
Inorganic nutrients including nitrogen largely influence on the growth and morphogenesis of plants. For example, in photosynthetic tissues of maize, enzymes related to C4 photosynthesis such as phosphoenolpyruvate carboxylase (PEPC) are synthesized and accumulated depending on the amount of inorganic nitrogen absorbed from the roots (B. Sugiharto, K. Miyata, H. Nakamoto, H. Sasakawa, T. Sugiyam, Plant Physiol. 92:963, 1990). Sugiharto et al. (B. Sugiharto, J. N. Burnell, T. Sugiyama, Plant Physiol. 100:153, 1992) and Suzuki et al. (I. Suzuki, C. Cretin, T. Omata, T. Sugiyama, Plant Physiol. 105:1223, 1994) discovered that this synthesis and accumulation are caused by the promotion of expression of C4Ppc1, a gene encoding phosphoenolpyruvate carboxylase (PEPC), by a cytokinin. Cytokinins are plant hormones synthesized in roots (L. J. Feldmn, in The Development and Function of Roots, J. G. Torrey and D. T. Clarkson Eds., Academic Press, London, 1975, pp.55-72), and thus it was found that they are important signal substances which transmit the information about the amount of the inorganic nitrogen, that was sensed by the roots.
When bacteria respond to various stimulations from outside, signal transduction mechanism called two-component signal transduction system plays an important role. It has become clear that the two-component signal transduction system is an important signal transduction system also in protozoa, fungi and plants (L. A. Alex and M. I. Simon, Trends Genet. 10:133, 1992). The two-component signal transduction system is composed of a sensor protein which senses stimulations in the outside, and a regulator protein which mediates the signal and causes various reactions. The signal transduction from the sensor protein to the regulator protein is mediated by phosphorylation reaction. The sensor protein serves as a kinase specific to the regulator protein, and the regulator protein is modified by the phosphorylation. The regulator protein is activated by phosphorylation and causes specific gene expression or the like. The sensor protein has a region in the N-terminal region, which senses the stimulation and a transmitter region in the C-terminal region, that phosphorylates the regulator protein. The regulator protein has a receiver region in the N-terminal region, which is phosphorylated, and a region in the C-terminal region, that has an enzyme activity, DNA-binding activity or the like for causing various reactions.
Known plant proteins related to the two-component signal transduction system include the following:
ETR1: This protein is a kind of receptor proteins of ethylene in Arabidopsis. This protein is a kind of sensor proteins and has a region homologous to receiver regions of regulator proteins (C. Chang, S. K. Kwok, A. B. Bleecker, E. M. Meyerowits, Science 262:539-544, 1993).
ERS: This protein is a kind of ethylene receptor proteins of Arabidopsis and a kind of sensor proteins. Its ethylene-receiving region and the transmitter region have high homologies to those of ETR 1. It does not contain a region homologous to the receiver region (J. Hua, C. Chang, Q. Sun, E. M. Meyerowitz, Science 269:1712-1714, 1995).
NR: This protein is an ethylene receptor protein of tomato. This protein has a high homology to ETRI but does not have a region homologous to the receiver region, like ERS (J. Wilkinson, M. B. Lanahan, H.-C. Yen, J. J. Giovannoni, H. J. Klee, Science 270:1807-1809, 1995).
CKI1: This protein is thought to be a cytokinin receptor protein of Arabidopsis and is a kind of sensor proteins. This protein has a region homologous to the receiver region like ETRI, in addition to the cytokinin receptor region and a transmitter region (T. Kakimoto, Science 274:982-985, 1996).
Thus, those hitherto known in plants are sensor proteins similar to the sensor proteins of microorganisms or sensor proteins which have regions homologous to the receiver region of a regulator protein. Thus, no plant regulator proteins are known.
Thus, it has been suggested that the signal transduction system in plants are composed of proteins and mechanisms having considerably different properties from those involved in the signal transduction system of microoganisms.
On the other hand, if a protein or a DNA encoding a protein in a plant is known, a protein resulting from modification of the protein may be expressed in plants, expression of the protein in the plant may be inhibited, the protein may be expressed in excess, or expression control of the protein may be modified by using recombinant DNA technology and the technology to construct transgenic plants.
Therefore, if a protein in a signal transduction system is known, manipulation of the signal tansduction system may be possible, and in turn, various life phenomena downstream thereof may be manipulated.
An object of the present invention is to provide a regulator protein in a two-component signal trnasduction system of a plant and a nucleic acid coding therefor.
The present inventors isolated a cDNA of a gene specifically induced by a cytokinin, from green leaves of maize in nitrogen-deficient state by using differential display method; sequenced the cDNA and determined the amino acid sequence of the protein encoded by the cDNA; discovered that the amino acid sequence has a homology to regulator proteins of microorganisms and that the amino acid sequence has characteristic amino acids common to regulator proteins of microorganisms; and
confirmed that the gene is induced by nitrogen or a cytokinin; thereby confirming that the protein is a regulator protein, to complete the present invention.
That is, the present invention provides an isolated protein having the amino acid sequence shown in SEQ ID NO:1 in the Sequence Listing or an amino acid sequence having a homology of not less than 30% to the amino acid sequence shown in SEQ ID NO:1, which functions as a regulator protein in a plant. The present invention also provides an isolated nucleic acid encoding the protein of the present invention.
Only sensor proteins are known as proteins involved in two-component signal transduction systems of plant cells. In monocotyledons, even a protein involved in a two-component signal transduction system is not known. Thus, by the present invention, a regulator protein of a two-component signal transduction system of plant cells and a nucleic acid coding therefor were first provided. Especially, a protein in a two-component signal transduction system in monocotyledon was first provided by the present invention. Thus, by the present invention, the signal transduction system of a cytokinin may be effectively modified by manipulation of the two-component signal transduction system so as to effectively control the various physiological mechanisms.